DE69233489T2 - Process for the preparation of diagnostics - Google Patents

Abstract

Microcapsules are prepared by a process comprising the steps of (i) spray-drying a solution or dispersion of a wall-forming material in order to obtain intermediate microcapsules and (ii) reducing the water-solubility of at least the outside of the intermediate microcapsules.Suitable wall-forming materials include proteins such as albumin and gelatin.The microcapsules have walls of 40-500 nm thick and are useful in ultrasonic imaging. The control of median size, size distribution and degree of insolubilisation and crosslinking of the wall-forming material allows novel microsphere preparations to be produced.

Description

The The present invention relates to the production of diagnostic agents, comprising hollow, preferably protein-containing, microcapsules suitable for reinforcement an ultrasound imaging are used.

The Fact that bubbles in the body can be used for echocardiography, is already some time known. Bubble-containing liquids can be injected into the bloodstream for this purpose (see Ophir et al. (1980) "Ultrasonic Imaging "2, 67-77, the Bubbles stabilized in a collagen membrane, US-A-4 446 442 (Schering) and EP-A-131 540 (Schering)) and EP-A-224 934 and EP-A-324 938 disclose the use of sonication produced by sonicating an albumin solution Blow. The size distribution the bubbles, however, are obviously not controllable and the bubbles disappear when they are pressurized, which they left in the left Ventricles (Shapiro et al., (1990) J. Am. Coll. 16 (7), 1603-1607).

The EP-A-52 575 discloses for the same use solid particles that are inside gas carry, wherein the gas is released from the particles in the bloodstream.

EP-458 745 (Sintetica) discloses a process for producing air or gas-filled Microballoons by interfacial polymerization synthetic polymers, for example polylactides and polyglycolides. WO 91/12823 (Delta) discloses a similar method using of albumin. Wheatley et al. (1990) Biomaterials 11, 713-717 ionotropic gelling of alginate to form microbubbles of one diameter from above 30 μm. The WO 91/09629 discloses liposomes for use as ultrasound contrast agents.

We found out now that a method of atomizing a solution of a Microcapsule-forming substance and the subsequent insolubilization of the formed Microcapsules leads to an improved product. Przyborowski et al. (1982, Eur. J. Nucl. Med. 7, 71-72) disclosed the preparation of microspheres of human serum albumin (HSA) by spray drying for one radioactive labeling and subsequent use in scintigraphic Illustration of the lung. It was not said that the microspheres are hollow, and it was at our repetition of work only solid microspheres generated. If the particles are not hollow, they are for echocardiography not suitable. Furthermore, the microspheres were in a one-step process from which we found that it was for the manufacture of for echocardiography suitable microcapsules not suitable; it was in the previous procedure necessary, to remove undenatured albumin from the microspheres not necessary for our procedure is); and, apparently, a wide size range of the microspheres received, as a further screening stage was necessary. Therefore was not only the method of Przyborowski et al. obviously none, that for the preparation of microcapsules useful in ultrasound imaging chosen but the manufactured particles were also for the purpose not suitable. We found a considerable one Improvement over the former Procedure out.

Of the Article by Przyborowski et al. refers to two earlier revelations of methods for forming albumin particles for pulmonary scintigraphy. Aldrich & Johnston (1974) Int. J. Appl. Rad. Isot. 25, 15-18 disclosed the use a spinning disk for producing particles of a Diameter of 3-70 microns, which then denatured in hot oil become. The oil is removed and the particles are labeled with radioisotopes. Raju et al. (1978) Isotope Practice 14 (2), 57-61 used the same Technique of a fast rotating disc, denatured the albumin however, by simply heating the particles. In no case were mentioned hollow microcapsules and the particles produced were not useful for echocardiography.

One Aspect of the present invention is the provision of a method this is a first stage of sputtering a solution or dispersion of a proteinaceous wall-forming material for Formation of pharmaceutically acceptable microcapsules by evaporation of the liquid carrier includes.

Preferably the product thus obtained becomes a second stage of reduction the water solubility from at least the outside subjected to the microcapsules.

The two levels can be carried out as a single process or the intermediate the first stage can be collected and separated in the second stage be treated. These two options will be in the following as the one-step process and the two-step process designated.

A second aspect is the provision of pharmaceutically acceptable microcapsules according to the Definition in claim 1 or claim 2.

The wall-forming material and the process conditions should be chosen so that the product is sufficiently non-toxic under the conditions of use and not immunogenic, which is clear from the dose administered and the duration of the treatment. The wall-forming material may be a starch derivative, a synthetic Polymer, such as tert-butyloxycarbonylmethylpolyglutamate (US-A-4 888 398) or a polysaccharide such as polydextrose.

In general, the wall-forming material may be selected from highly hydrophilic, biodegradable, physiologically compatible polymers. Of such polymers, low water solubility polysaccharides, polylactides and polyglycolides and their copolymers, copolymers of lactides and lactones such as ε-caprolactone, δ-valerolactone, polypeptides and proteins such as gelatin, collagen, globulins and albumins may be cited. Other suitable polymers include poly (ortho) esters (see, for example, US-A-4,093,709, US-A-4,131,648, US-A-4,138,344, US-A-4,180,646, polylactic and poly glycolic acid, and US Pat their copolymers, for example DEXON (see J. Heller (1980) Biomaterials 1.51), poly (DL-lactide-co-δ-caprolactone), poly (DL-lactide-co-δ-valerolactone), poly (DL-lactide poly (β-aminoketones (Polymer 23 (1982), 1693); polyphosphazenes (Science 193 (1976), 1214); and polyanhydrides degradable polymers can be found in R. Langer et al., (1983) Macromol. Chem. Phys., C23, 61-125. Polyamino acids, such as polyglutamic and polyaspartic acid, can also be used, as well as their derivatives, ie partial esters with lower alcohols or glycols A useful example of such polymers is poly (tert-butylglutamate) copolymers with other amino acids, for example, methionine, leucine Valine, proline, glycine, alanine and the like are also possible. Recently, some novel derivatives of polyglutamine and polyaspartic acid with controlled biodegradability have been reported. (See WO 87/03891; U.S. 4,888,398 and EP-130,935). These polymers (and copolymers with other amino acids) have formulas of the following type: - (NH-CHA-CO) _x (NH-CHX-CO) _y wherein X denotes the side chain of an amino acid residue and A is a group of the formula - (CH ₂ ) _n COOR ¹ R ² OCOR (II) wherein R ¹ and R ^{2 are} H or lower alkyl and R is alkyl or aryl or R and R ¹ are linked together by a substituted or unsubstituted linking member to form a 5- or 6-membered ring.

A can also be used for groups of formulas - (CH ₂ ) _n COO-CHR ¹ COOR (I) and - (CH ₂ ) _n CO (NH-CHX-CO) _m NH-CH (COOH) - (CH ₂ ) _p COOH (III) and corresponding anhydrides. In all of these formulas, n, m and p are smaller integers (not more than 5) and x and y are also integers chosen so that the molecular weights are not less than 5,000.

The polymers mentioned are suitable for the preparation of the microspheres according to the invention and, depending on the nature of the substituents R, R ¹ , R ² and X, the properties of the wall, for example the strength, elasticity and biodegradability, can be controlled. X can be, for example, methyl (alanine), isopropyl (valine), isobutyl (leucine and isoleucine) or benzyl (phenylalanine).

Conveniently, the wall-forming material is proteinaceous. For example, can It is preferably collagen, gelatin or (serum) albumin, in any case of human origin (i.e., derived from or derived from human Protein of appropriate nature). Preferably, it is human serum albumin (HA), that from blood donations or ideally from the fermentation of microorganisms (including Cell lines), which for an expression of HA has been transformed or transfected.

Methods of expressing HA (which term includes analogs and fragments of human albumin, such as those of EP-A-322 094, and polymers of monomeric albumin) are disclosed, for example, in EP-A-201 239 and EP-A-286 424. "Analogs and fragments" of HA encompass all peptides which (i) can form a microcapsule in the method of the invention and (ii) of which a continuous Re gion of at least 50% (preferably at least 75%, 80%, 90% or 95%) of the amino acid sequence is at least 80% homologous (preferably at least 90%, 95% or 99% homologous) to a contiguous region of at least 50% ( preferably 75%, 80%, 90% or 95%) of human albumin. HA produced by recombinant DNA techniques is particularly preferred. Therefore, the HA can be generated by expressing a nucleotide sequence coding for HA in yeast or other microorganism and purifying the product according to the relevant known methods. Such a material lacks the fatty acids associated with serum-derived material. Preferably, the HA is substantially free of fatty acids; ie it contains less than 1% of the fatty acid concentration of serum-derived material. Preferably, fatty acid is not detectable in the HA.

In In the following description of preferred embodiments, the term "protein" is used as we this, however, can Of course, as discussed above, other biologically compatible wall-forming Materials are used.

The protein solution or dispersion is expediently 0.1-50% strength (W / v), preferably about 5.0-25.0% to protein, especially if the protein is albumin. About 20% is optimal. It can Mixtures of wall-forming materials are used, wherein in this case the percentages in the last two sentences refer to the total content of wall-forming materials.

The to be sprayed Preparation can except the wall-forming material and solvent or carrier liquid contain other substances. Therefore, the aqueous phase can be 1-20 wt%. water-soluble hydrophilic Compounds, such as sugars and polymers, as stabilizers, for example Polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), gelatin, polyglutamic acid and polysaccharides, such as starch, Dextran, agar, xanthan and the like. Similar aqueous phases can be used as the carrier liquid used in which the final microsphere product before use is suspended. It can emulsifiers be used (0.1-5 Wt .-%), the physiologically very acceptable Emulsifiers include, for example, egg lecithin or soybean lecithin, or synthetic lecithins, such as saturated synthetic lecithins, for example, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine, or unsaturated synthetic lecithins, such as dioleylphosphatidylcholine or dilinoleylphosphatidylcholine. Emulsifiers can also wetting agents, such as free fatty acids, Esters of fatty acids with polyoxyalkylene compounds such as polyoxypropylene glycol and polyoxyethylene glycol; Ethers of fatty alcohols with polyoxyalkylene glycols; Esters of fatty acids with polyoxyalkylated sorbitan; Soap; Glycerol polyalkylenstearat; Glycerol-polyoxyethylene ricinoleate; Homo- and copolymers of polyalkylene glycols; polyethoxylated soybean oil and castor oil and hydrogenated derivatives; Ethers and esters of sucrose or others Carbohydrates with fatty acids, Fatty alcohols, these optionally being polyoxyalkylated; Mono-, Di- and triglycerides of saturated or unsaturated fatty acids, Glycerides or soybean oil and sucrose.

Additions can be made in the wall of the microspheres incorporated into the physical properties, such as dispersibility, elasticity and water permeability, to modify.

From the useful additives you can lead connections, which can "hydrophobize" the wall the water permeability to reduce, for example, fats, waxes and hydrocarbons high Molecular weight. Additions, the dispersibility of the microspheres in the injectable Improve fluid carrier, are amphipathic compounds, such as the phospholipids; they increase too the water permeability and the biodegradation rate.

Additives that the elasticity of change increase, are plasticizers such as isopropyl myristate and the like. Also very useful additives consist of polymers with the the wall itself are related, but a relatively low molecular weight exhibit. For example, you can when using copolymers of the polylactic acid / polyglycolic acid type as wall-forming material, the properties of the wall in an advantageous Be modified (increased Softness and biodegradability) by adding as additives Low Molecular Weight Polyglycolides or Polylactides (1000-15000 daltons) be incorporated. Also, polyethylene glycol of a moderate to low molecular weight (eg PEG 2000) is a useful one Plasticizer additive.

The Amount of additives to be incorporated into the wall is extremely variable and hang from the respective need. In some cases, no additive is used at all; in other cases are amounts of additives that reach about 20% by weight of the wall can, possible.

The protein solution or dispersion hereinafter referred to as the "protein preparation" (preferably solution) is atomized and spray dried by any suitable technique resulting in discrete microcapsules of 1.00-50.0 μm in diameter. These numbers refer to at least 90% of the population of microcapsules whose diameter is measured with a Coulter Master Sizer II. The term "microcapsules" means hollow particles enclosing a space, the space being filled with a gas or vapor, but not with any solid materials. Honeycomb particles resembling confectionery sold in the United Kingdom as "Maltesers" (registered trademark) are not formed. It is not necessary that the space be completely enclosed (although this is preferred), and it is not necessary for the microcapsules to be exactly spherical, although they are generally spherical. When the microcapsules are non-spherical, the diameters given above refer to the diameter of a corresponding spherical microcapsule which has the same mass as compared to the non-spherical microcapsule and which comprises the same volume of the void.

The atomization comprises the formation of an aerosol of the protein preparation by, for example, driving through the preparation by at least one opening under pressure in one Chamber with warm air or other inert gas or through Use of a centrifugal atomizer in a corresponding chamber. The chamber should be big enough to be the largest expelled droplet not on the walls meet before they are dried. The gas or the steam in the Chamber is clean (i.e. preferably sterile and pyrogen free) and non-toxic if it (he) in the bloodstream in the with the administration the microcapsules related Amounts used in echocardiography. The evaporation rate the liquid from the protein preparation should be sufficiently high that hollow Microcapsules are formed, but not so high that the Microcapsules burst. The evaporation rate can be adjusted by varying the Gas flow rate, the protein concentration in the protein preparation, the nature of the protein Liquid carrier, the Addition rate of the solution and what of highest importance is the Temperature of the gas, which meets the aerosol, are controlled. At an albumin concentration of 15-25% in water is an inlet gas temperature of at least about 100 ° C, preferably at least 110 ° C in general to ensure the hollowness sufficient and the Temperature can reach 250 ° C be high without the capsules bursting. About 180-240 ° C, conveniently about 210-230 ° C and preferably about 220 ° C is at least for Albumin optimal. The temperature can be in the single level version of the inventive method be sufficient to at least a part (usually the outside) of the wall-forming material and often practically the whole wall-forming material insoluble close. As the temperature of the gas that hits the aerosol, also on the delivery rate of the aerosol and on the liquid content of the protein preparation depends can the outlet temperature to ensure adequate Temperature to be monitored in the chamber. An outlet temperature of 40-150 ° C proved to be suitable. Apart from this factor, however, proved the control of the flow rate as not as useful as the control of the other parameters.

in the Two-step process typically involves the intermediate microcapsules 96-98% monomeric HA and they have a limited lifespan in vivo for Ultrasound imaging. You can however, they can be used for ultrasound imaging or they can be stored and before the implementation the second stage of the two-stage process are transported. They therefore form a further aspect of the invention.

In the second stage of the process will be those in the first stage produced intermediates Microcapsules fixed and made less water soluble, so they longer Time remain, whereby they are not so insoluble and inert that they are not biodegradable. This level also strengthens the Microcapsules, allowing them to avoid the rigors of administration, vascular thrust and Ventricular pressure, better able to withstand. If the microcapsules burst, they are less echogenic. Schneider et al. (1992) Invest. Radiol. 27, 134-139 showed that sound-treated albumin microbubbles of the prior art did not have this strength and their echogenicity was rapid lost when she's for the left ventricle were exposed to typical pressure values. In In the second stage of the process, heat (for example, microwave heat, radiant heat or name is Air, for example, in a usual Oven), ionizing radiation (with, for example, a dose of gamma radiation from 10.0-100.0 kGy) or chemical crosslinking using, for example Formaldehyde, glutaraldehyde, ethylene oxide or other means for Crosslinking of proteins will be applied and become the second stage on the practically dry intermediate microcapsules formed in the first stage or on a suspension of such microcapsules in a liquid, in which the microcapsules are insoluble are, for example, a suitable solvent carried out. In the one-step version of the process may be a cross-linking agent, like glutaraldehyde, into the spray-drying chamber sprayed or in the protein preparation immediately upstream of the sprayer introduced become. Alternatively, the temperature in the chamber may be high enough be insoluble to the microcapsules close.

The final product, measured in the same way as the intermediate microcapsules, can microcapsules having a diameter of 0.05-50.0 μm may be used as desired, but with the method according to the invention ranges of 0.1-20.0 μm and in particular 1.0-8.0 μm can be achieved and are preferred for echocardiography , We found that a range of about 0.5-3.0 microns is particularly suitable for producing a low-contrast image and for use in color Doppler imaging, with a range of about 4.0-6.0 microns for the He can be better at producing sharp pictures. In determining the size generated in the first stage, one must consider the fact that the second stage can change the size of the microcapsules.

It was found that the process according to the invention can be controlled in order to obtain microspheres with desired properties. Thus, the pressure at which the protein solution is supplied to the spray nozzle can be varied, for example from 1.0-10.0 x 10 ⁵ Pa, advantageously 2.0-6.0 x 10 ⁵ Pa, and preferably about 5 x 10 ⁵ Pa. Other parameters may be varied as disclosed above and below. In this way, novel microspheres can be obtained.

The Invention provides pharmaceutically acceptable hollow microspheres ready in the form of a dry powder in which more than 30%, preferably more than 40%, 50% or 60% of the microspheres a diameter within a 2 μm range and at least 90%, preferably at least 95% or 99% of a diameter within the range 1.0-8.0 μm.

Of the Interquartile range can therefore 2 microns with a median diameter of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5 μm be.

Therefore can at least 30%, 40%, 50% or 60% of the microbead diameter within of the range 1.5-3.5 μm, 2.0-4.0 μm, 3.0-5.0 μm, 4.0-6.0 μm, 5.0-7.0 μm or 6.0 -8.0 μm. Preferably, this percentage of microspheres has a diameter within a 1.0 μm range, for example 1.5-2.5 μm, 2.0-3.0 μm, 3.0-4.0 μm, 4.0-5.0 μm, 5.0-6.0 μm, 6.0 μm 7.0 μm or 7.0-8.0 μm, on.

The microspheres can proteinaceous walls wherein at least 90%, preferably at least 95% or 99% of the microspheres have a diameter in the range 1.0-8.0 microns; at least 90%, preferably at least 95% or 99% of the microspheres have a wall thickness of 40-500 nm, preferably 100-500 nm and at least 50% of the protein in the walls of the microspheres is networked. Preferably, at least 75%, 90%, 95%, 98.0%, 98.5% or 99% of the protein is sufficiently cross-linked to an extraction with a 1% HCl solution 2 minutes to be able to withstand. Extracted protein is extracted using the Coomassie blue protein assay, Bradford, demonstrated. The degree of crosslinking is determined by varying the heating, the Irradiation or chemical treatment of the protein. While In the crosslinking process, protein monomer is crosslinked and it becomes quickly for a simple dissolution process not available, which is detected by gel permeation HPLC or gel electrophoresis as indicated in Example 8 below. A continued treatment leads to a further networking of already cross-linked material, so that it for the HCl extraction described above is no longer available. While heating at 175 ° C lose rHA microspheres according to the invention about 99% of HCl extractable protein over 20 minutes, while heating at 150 ° C while Removed only about 5% HCl-extractable protein for 20 minutes and for 30 min 47.5% while 40 min 83%, while 60 min 93% while 80 min 97% and during 100 min 97.8% of the HCl-extractable Protein removed. Therefore, to get good levels of networking, can the microspheres at 175 ° C at least 17-20 minutes long, at 150 ° C for at least 80 minutes and at other temperatures while appropriate longer or shorter Times are heated.

At least 10% of the microspheres, after suspending in water, can withstand the application of a pressure of 2.66 × 10 ⁴ Pa for 0.25 seconds without bursting, collapsing or filling with water. The transient maximum pressure in the human left ventricle is about 200 mm Hg (2.66 x 10 ⁴ Pa). Preferably, 50%, 75%, 90% or 100% survive the 0.26 second application of 2.66 x 10 ⁴ Pa in the above assay, ie they remain echogenic. In vivo, preferably, the same percentages remain echogenic during passage through both ventricles of the heart.

The injectable microspheres of the present invention can be stored dry in the presence or in the absence of additives to improve preservation and prevent coalescence. As additives may be from 0.1-25 wt .-% of water-soluble physiologically acceptable compounds such as mannitol, galactose, lactose or sucrose, or hydrophilic polymers such as dextran, xanthan, agar, starch, PVP, polyglutamic acid, polyvinyl alcohol ( PVA) and gelatin. The useful life of the microspheres in the injectable fluid carrier phase, ie Span while observing useful echo signals may be controlled to take from a few minutes to several months, depending on demand; this can be done by controlling the porosity, solubility or degree of crosslinking of the wall. These parameters can be controlled by proper selection of the wall-forming materials and additives and by adjusting the rate of evaporation and the temperature in the spray-drying chamber.

to Minimization of agglomeration of the microcapsules can Microcapsules with a suitable inert diluent using a Fritsch centrifugal pin mill equipped with a 0.5 mm sieve or Milling a Glen Creston air impact jet mill become. Suitable extenders are finely ground powders which are inert and to the intravenous Are suitable, such as lactose, glucose, mannitol, sorbitol, galactose, Maltose or sodium chloride. Once the microcapsules / extender mixture ground, it can be superseded in an aqueous medium, around the removal of non-functional / faulty To facilitate microcapsules. In the restoration in the aqueous phase is it cheap a trace amount of a wetting agent to prevent agglomeration to use with. For anionic, cationic and nonionic suitable for this purpose Wetting agents include poloxamers, sorbitan esters, polysorbates and lecithin.

The Microcapsule suspension can then be allowed to float or be centrifuged to detect defective particles with surface defects, when filling a filling with liquid and a loss of echogenicity would cause to sediment.

The Microcapsule suspension can then be used to ensure a uniform particle distribution remixed, washed and in for intravenous injection suitable buffer, for example, 0.15 M NaCl, 0.01 mM Tris, pH 7.0, to be restored. The suspension may be in aliquots for freeze-drying and subsequent sterilization by For example, gamma radiation, dry heating or ethylene oxide be split.

One alternative method for deagglomeration of the insolubilized or fixed microcapsules is their direct suspension in one aqueous medium, that is a wetting agent, selected from poloxamers, sorbitan esters, polysorbates and lecithin. The Deagglomeration can then be carried out using a suitable homogenizer be achieved.

The Microcapsule suspension can then be allowed to float or be centrifuged to the faulty particles as above to sediment, and it can further as above be treated.

Even though the microspheres of this invention can be marketed in the dry state, in particular when designed with a limited life after injection may be desired even finished preparations, i. ready for injection Suspensions of microspheres in an aqueous liquid carrier, too to sell.

However, the product is generally supplied and stored as a dry powder and is suspended in a suitable sterile non-pyrogenic liquid immediately prior to administration. The suspension is generally administered by injection of about 1.0-10.0 ml into a suitable vein, for example the forearm vein or another blood vessel. A microcapsule concentration of about 1.0 x 10 ⁵ to 1.0 x 10 ¹² , preferably about 5.0 x 10 ⁵ to 5.0 x 10 ⁹ particles / ml is suitable.

Even though Ultrasound imaging on the most diverse animal and human Body organ systems applicable One of their main applications is to take pictures of myocardial tissue and perfusion or blood flow patterns.

at The method is one of a scanner and an imaging device existing ultrasound scanner used. The device creates visible images of a given area, in this Fall of the heart region of a human body. Typically will the transducer directly on the skin over the area to be imaged placed. The scanner includes various electronic components including ultrasonic transducers. The transducer generates ultrasonic waves that are a sector scan perform the heart region. The ultrasound waves are reflected by the different parts of the heart region and received by the receiving transducer and known accordingly relevant Pulsechover processes. After processing, the Signals to the (also known in the art) imaging device sent for viewing.

at The method of the present invention is described after the "preparation" of the patient and the presence of the scanner in place, the microcapsule suspension injected, for example by a brachial vein. The contrast agent flows through the vein to the right venous Side of the heart, through the main pulmonary artery leading to the lungs, through the lungs, through the capillaries, into the pulmonary vein and after all in the left atrium and the left ventricular cavity of the heart.

With The microcapsules of this invention can be observed and diagnosed in terms of the for the blood needed to pass the lungs, blood flow patterns, the size of the left Atria, the final ability the mitral valve (the left atrium and the left ventricle separates), chamber dimensions in the left ventricular cavity and Wandbewegungsabnormitäten be made. When expelling the contrast agent from the left Ventricle can be the final ability the aortic valve are also analyzed as well as the Austreibanteil or percentage of the volume expelled from the left ventricle. Finally give the contrast patterns in the tissue, which areas may not adequately perfused become.

In summary, can such a pattern of pictures makes the diagnosis more unusual Blood flow characteristics in the heart, the ability to close the valves, chamber sizes and Support wall movement and a possible Indicator for deliver myocardial perfusion.

The Microcapsules can starting from intravenous Injections allow imaging of the left heart. The albumin microcapsules can when injected into a peripheral vein to a transpulmonary passage be able. this leads to on echocardiographic opacification of the left ventricle (LV) chamber as well as myocardial tissue.

Next The scanner described shortly above has others Ultrasonic scanners, for what Examples are disclosed in U.S. Patent Nos. 4,134,554 and 4,315,435, the disclosures of which are incorporated herein by reference Revelations are hereby incorporated by reference. Basically refer These patents apply to various procedures including cross-sectional dynamic echography (DCE) for the production of sequential two-dimensional images of cross-sectional sections of an animal or human anatomy using ultrasound energy at a frame rate, the to enable the dynamic visualization of moving organs sufficiently is. Device types used with DCE become common referred to as a DCE scanner and they transmit and receive short Sound pulses in the form of narrow beams or lines. The strength of reflected signals is a function of time using a nominal Schallge speed converted into a location is and on a cathode ray tube or other appropriate Devices in an analogous to radar or sonar devices Way is displayed. While DCE for generating images of many organ systems including liver, Gallbladder, pancreas and kidney, it is often used to visualize Tissue and main blood vessels of the Used heart.

The Mirocapsules can for imaging a large Variety of areas are used, even if they are at one peripheral venous To be injected. These areas include (without limitation): (1) the venous Drainage system to the heart; (2) the myocardial tissue and perfusion characteristics while an exercise load test or the like, and (3) myocardial tissue after oral administration or intravenous injection of medicines designed to increase blood flow to the tissue. Furthermore can the microcapsules to track changes in the blood circulation of myocardial tissue due to surgery, such as (1) vein grafting the coronary artery; (2) Coronary artery angioplasty (balloon dilatation of a coronary artery) narrowed artery); (3) Use of thrombolytic agents (such as Streptokinase) for dissolution clots in coronary arteries or (4) circulatory defects or changes due to a past Heart attacks are suitable.

Further can at the time of a coronary angiogram (or a digital Subtraction angiogram) an injection of the microcapsules data in terms of provide tissue perfusion properties comparable to those of the angiogram measure, which only identifies the anatomy of the cardiac vessels Improve and complete data can.

By the use of the microcapsules of the present invention may be other non-cardiac organ systems, including the liver, spleen and of the kidneys currently being imaged by ultrasound techniques to improve such currently obtainable images and / or Generate new images that improve blood circulation and flow characteristics show previously using ultrasound imaging techniques the prior art were not accessible to a figure, suitable.

Preferred aspects of the present invention will hereinafter be described by way of example and under Be Train described on the pictures, wherein

1 is a partially cut away perspective view of a suitable spray drying apparatus for the first stage of the method according to the invention from the front and from one side,

2 Fig. 2 is a graph showing how the degree of fixation of the microbead walls (in this case albumin) can be controlled by varying the temperature and duration of heating in the second stage of the process, and

3 Fig. 2 is a graph showing how the pressure resistance of the microspheres can be varied by changing the length of the heating time in the second stage of the process.

example 1

A suitable spray dryer ( 1 ) is available from A / S Niro Atomizer, Soeborg, Denmark under the trade name "Mobile Minor". Details of its construction are given immediately before the claims. It comprises a centrifugal atomiser (type M-02 / B Minor), which is driven by an air turbine with an air pressure of a minimum of 4 bar and a maximum of 6 bar. At 6 bar, a Zerstäuberradgeschwindigkeit of about 33,000 min ^{-1 is} achieved. The switching on and off of the compressed air to the atomizer by means of a valve placed in the instrument panel. The maximum consumption of compressed air to the atomizer is 17 Nm ³ / h at a pressure of 6 bar. All parts that come into contact with the liquid feed and powder are made of AISI 316 stainless steel except for the pump feed tube and atomizer wheel, which is made of AISI 329 stainless steel to withstand high centrifugal forces.

The Drying chamber has a made of stainless steel AISI 316 Inside well insulated with Rockwool and outside with a mild steel coating is covered. The drying chamber is equipped with a sidelight and observation glass for inspection during of the operation. The roof of the drying chamber is inside made of stainless steel AISI 316 and outside of stainless steel AISI 304th

One stainless steel AISI 304 existing air distributor becomes the Distribution of the air in the drying chamber used to the best possible Drying effect to achieve. One made of stainless steel AISI 316 Air line ensures a lateral transport of the exhaust air and the powder to the cyclone, made of stainless steel AISI 316 and the powder and the Air should separate.

One closing valve throttle valve type, which is also made of AISI 316 stainless steel and a seal made of silicone rubber is for discharging of the powder under the cyclone into one by means of a spring device firmly used under the cyclone powder collecting glass crucible used.

One Silumin blower with a three-phase short-circuit motor of 0.25 kW and V belt drive with belt monitoring draws air and powder through the drying chamber and the cyclone.

A Air heater heated the drying air by means of electricity (total consumption 7.5 kWh / h, unlimited adjustable) and can intake air temperatures up to about 350 ° C although this is generally too high for the preparation of the microcapsules of the invention is.

A two-fluid nozzle sputtering apparatus made of AISI 316 stainless steel and consisting of an inlet conduit with a nozzle support and a nozzle may be added and placed on the ceiling of the drying chamber. The apparatus includes an oil / water separator, a reduction valve and a compressed air pressure valve to the two-fluid nozzle. Consumption of compressed air: 8-15 kg / h at a pressure of 0.5-2.0 bar (0.5-2.0 × 10 ⁵ Pa).

A suitable pump for transporting the feed material of the wall-forming preparation to the sputtering device is a peristaltic pump. The pump is equipped with a motor (1 × 220 V, 50 Hz, 0.18 kW) and a continuously adjustable gearbox for equipped with manual adjustment. One from a silicone hose existing feed line leads from a conveyor tank (local supply) through the feed pump to the atomizing device.

An anhydrous air filter composed of a pre-filter, a stainless steel filter housing and an anhydrous air filter is used to treat the incoming drying air with these completely pure.

A 20% solution of sterile pyrogen-free rHA in pyrogen-free water (for injection) to the nozzle a two-fluid nozzle atomizing device, those in the commercial described above Spray drying unit was mounted, pumped. The speed of the peristaltic pump was maintained at a rate of about 10 ml / min inlet temperature of 220 ° C the outlet temperature at 95 ° C was held.

The two-fluid atomization nozzle was supplied with compressed air at 2.0-6.0 bar (2.0-6.0 × 10 ⁵ Pa). In this area microcapsules are obtained with a mean size of 4.25-6.2 microns.

typically, led an increase in mean particle size (due to reduced atomization pressure) to an increase in the amount of microcapsules over 10 μm in size (see Table 1).

Table 1 Effects of atomization pressure on the frequency of microcapsules of diameter greater than 10 μm

In the second stage of the process, 5 grams of microcapsules were heated in a glass beaker using a Gallenkamp forced air oven. A temperature of 175 ° C for 1 h was sufficient to provide microcapsules with 100% fixation as determined by HPLC. The effect of this heat fixation was to increase the echogenic half-life in vitro from a few seconds to more than 30 minutes. By changing the temperature and length of incubation, it is possible to vary the degree of fixation between about 5% and 100%. Examples of heat fixing profiles at different temperatures are in 2 specified.

After heat-setting, the microcapsules were deagglomerated in one of two ways and dispersed in water. Method 1 involved initially mixing the heat-set beads with an equal weight of finely ground lactose (average diameter: 5 μm). The mixture was then run through a Fritsch centrifuge mill with a 0.5 mm screen and a 12 tooth rotor. The ground beads were collected and run through the mill a second time to ensure that a complete mix had occurred. The milled powder was then resuspended in water containing 1 mg.ml ^{-1 of} Pluronic F68. Typically, 10 grams of microcapsules and lactose were added to 100 ml of water and Pluronic F68. The deagglomeration method 2 comprises adding 5 g of the heat-fixed microcapsules to 100 ml of water containing 100 mg of Pluronic F68. The microcapsules were dispersed using a Silverson homogenizer (Model L4R with a 2.54 cm tubular homogenizing head and a high shear sieve) and homogenized for 60 seconds.

The resuspended globules were transformed into intact (gas-containing) and broken globules using a Flotation technology separated. The gassy globules floated over one Time span of 1 h visible to the surface and were from the sinking fraction, which does not contain the required gas, decanted off. The separation process can be accelerated by centrifuging. A 30 second Centrifugation with 5000 × g is sufficient to separate the two fractions.

After separation, the intact microcapsules were lyophilized in the presence of lactose and Pluronic F68. Optimum conditions for freeze-drying included resuspending 30 mg of microcapsules in 5 ml of water containing 50 mg lactose and 50 mg Pluronic F68. The freeze-dried microcapsules may be in a liquid (eg, water, saline) to form a monodisperse redistribution.

Example 2

The procedure of Example 1 was repeated, but with the following differences in the first stage: a centrifugal atomizing device was used instead of a two-fluid nozzle; the inlet temperature was 150 ° C (with the outlet temperature still maintained at 105 ° C); and compressed air was supplied to the nozzle at 1.0-6.0 × 10 ⁵ Pa. The wheel rotated at 20-40000 min ^-1 yielding droplets and finally microcapsules with a number average diameter in the 1.0-8.0 μm range.

Example 3

In The second step of the process of Example 1 or 2 was as follows changed. A small aliquot of the microcapsules (0.5 g) was placed in a Microwave oven heated so that it uses 300-350 Wh microwave heat 2500 mHz received. This resulted in microcapsules in which 90-95% of the monomeric rHA were insoluble (which was determined by gel permeation chromatography), and as Result of this heat fixation took their echogenic half-life in vitro of a few seconds on over 30 min to.

Example 4

In the second step of the process of Example 1 or 2, the following was varied. A small aliquot of the microcapsules (0.5 g) was sealed under argon in a glass vial. The vial was cooled to 4 ° C and then irradiated with a ⁶⁰ Co gamma radiation source to provide a gamma ray dose of 15.0 kGy. Irradiation resulted in the formation of microcapsules in which 10-15% of the monomeric albumin was insoluble.

Example 5

In the second step of the process of Example 1 or 2, the following was varied. A small aliquot of the microcapsules (0.5 g) was enclosed under argon in a glass vial. The vial was cooled to 4 ° C and then irradiated with a ⁶⁰ Co gamma radiation source to provide a gamma ray dose of 50.0 kGy to the microcapsules. After irradiation, the microcapsules were incubated in oxygen at 50 ° C for 6 hours. Irradiation resulted in the formation of microcapsules in which 50-60% of the monomeric rHA was insoluble.

Example 6

In The second step of the process of Example 1 or 2 was as follows varied. A small aliquot of the microcapsules (0.5 g) was added in 5 ml of ethanol, chloroform or methylene chloride containing: (a) 1,5% glutaraldehyde, b) 2.0% diphthaloyl chloride or c) 5.0% formaldehyde, resuspended. The microcapsules were used for different periods of time stirred for 10 minutes to 3 hours. The microcapsules were removed by filtration and carefully placed in the original organic buffer containing 5% ethanolamine washed to excess cross-linking agent to remove. After all For example, the microcapsules were washed in an organic solvent and placed under Vacuum dried to remove any remaining solvents. Of the Degree of insolubility can be varied by this method from 5-100%, resulting in renewal echogenic half-life in vitro ranges from 1-2 min to more than 1 h.

Example 7

The two independent Stages of formation of microcapsules and insolubilization of the shell may occur in be combined into a single process. In this example will the formation of the microcapsules and the insolubilization of the polymeric material simultaneously during the spray drying process reached.

A solution of rHA was fed through a peristaltic pump to a small reaction chamber, with a separate feed line feeding a 5% solution of a suitable crosslinking agent, eg glutaraldehyde, diphthaloyl chloride or formaldehyde. The residence time in the reaction chamber was such that initial adduct formation between the crosslinking agent and the protein was achieved but cross-linking between proteins was prevented. The reaction vessel exit was directly to the two-fluid nozzle atomization devices, which in a specially designed spray-drying unit with the ability to handle volatile solvents were mounted. The conditions of spray drying were as outlined in Example 1. The microcapsules were incubated dry at room temperature to allow the formation of crosslinks between the proteins and then suspended in ethanol containing 5% ethanolamine to quench any remaining crosslinking agent. The microcapsules were washed thoroughly and finally dried under vacuum to remove residual solvent.

Example 8: Test for Free monomeric rHA in microcapsules

1 ml volume of ethanol was added to 100 mg microcapsules in a 20 ml glass bottle and sonicated for 30 seconds. To this suspension was added 19 ml of H ₂ O.

The Mixture was centrifuged in a tabletop microcentrifuge (Gilson) for 20 sec and the clear fraction was tested. The test was performed by 50 ml of the fraction is automatically loaded on a Shimadzu LC6A HPLC and on a TSK gel permeation column with a flow rate of 1 ml / min using sodium phosphate buffer (pH 7.0) were chromatographed.

The the rHA monomer peak levels were recorded and for determining the concentration of the monomer using a Standard curve between 1 and 10 mg / ml monomeric rHA used.

The percentage of free monomeric rHA was calculated by measuring the monomer concentration in the fixed microcapsules and indicating this number as a percentage of the monomer concentration of unfixed microcapsules. The results are in 2 specified.

Heating the spray-dried microcapsules in an oven (as described in Example 1) results in a decrease in the amount of monomer that can be detected ( 2 ). This decrease in the detectable monomeric rHA is due to the denaturation and crosslinking of monomeric rHA to insoluble polymers that can not be tested by the aforementioned HPLC method.

Using the HPLC method for testing rHA monitoring concentrations is off 2 it can be seen that after a 15-minute incubation in the rHA microcapsules no free monomeric rHA is present. However, it is still possible to further crosslink the rHA microcapsules by prolonged heating.

This prolonged Heating leads to an increased Degree of microcapsule crosslinking, which in turn increased microcapsules Strength generated, the corresponding more resistant to pressure are.

By careful Control of temperature and incubation time can be controlled with a microcapsule Range of crosslinking (and thus pressure resistance) are produced.

Example 9: Effects the incubation period at 175 ° C on the pressure resistance of rHA microcapsules

A batch of rHA microcapsules was divided into aliquots of 5 g and stored at 175 ° C for different times as in 3 shown fired.

Afterwards the heat fixation The amount of free monomer was as described in Example 8 certainly. For each of the incubations showed no monomeric rHA detectable.

The heat-set microcapsules were disaggregated using a Fritsch centrifugal mill (as described above) and the intact air-containing microcapsules were recovered by the flotation technique mentioned. The recovered microcapsules were suspended in water containing Pluronic F68 (1 mg / ml) at a concentration of 0.5 × 10 ⁸ capsules / ml. The resuspended air-containing microcapsules were exposed to elevated atmospheric pressure by applying pressure with a 50 ml syringe while this suspension was contained in a closed container (25 ml polystyrene container).

For each of the pressure values tested, the individual microcapsule suspension was exposed to the selected pressure and held at that pressure for 5 seconds before the pressure was released. For each suspension analyzed, the pressure increase was performed three times. The pressure in the closed Behäl ter was tested by an RS handheld manometer.

To In the pressure treatment, the microcapsule suspensions were examined by light microscopy and image analysis tested and the percentage of air / non-air Microcapsules determined. This analysis is performed because only the air-containing microcapsules the function of strengthening the Have ultrasound echo contrast.

Out 3 It can be seen that microcapsules fixed at 175 ° C for 60 minutes as described in Example 1 are stable at all pressures to which they were exposed in this experiment.

By careful Control the incubation period at this particular temperature (175 ° C) can microcapsule lots be prepared with different degrees of cross-linking, in turn, opposite different levels of pressure increase are stable.

By Use of this careful Control of cross-linking by adjusting the incubation time and temperature can Batches of air-containing microcapsules are produced that are specific are designed to withstand a designated pressure increase can.

The Temperature used to crosslink the microcapsules may as well like the length be varied indefinitely during the incubation period.

Example 10: Microcapsule Classification

One Advantage of the method according to the invention is that the mean size and size distribution the microspheres can be controlled. However, one can also desire desired sizes, for example by flotation choose. In a homogeneous dispersion of microspheres, larger particles rise due to the lower density (more encapsulated air) of the larger particles faster than smaller particles to the surface. Therefore, changes if you leave the dispersion, the particle size distribution in every height the solution in terms of time.

microcapsules were in 2000 ml of a 6% (w / v) sodium chloride and 0.1% (w / v) Pluronic Aqueous solution containing F68 in a glass bottle dispersed, wherein a liquid column of about 165 mm resulted. A sample tube became 50 mm below the surface the liquid placed to take samples at intervals.

By change The service life and sodium chloride concentration could be a variety of particle size distributions produced and microcapsules down to 2 microns downgraded classified.

Other wet techniques for classification include hydrodynamic chromatography and field flow fractionation. "Dry" techniques that the Principles of the slurry and cross-flow separation are commercially available in the form of microsplit (British Rem.) -, Zig-zag (Alpine) and Turbo (Nissuin) classifiers available. The angular flow classifier manufactured by Nitettsu Mining Co. uses a different principle (the Coanda effect), with also good results in the classification of microcapsules could be achieved.

Further details of the construction the sputtering device

Gewicht und Abmessungen Gewicht 280 kg Länge 1800 mm Höhe 2200 mm Tiefe 925 mm In 1 denotes the reference number 1 the feeder. 2 is a ceiling air dispersing device that ensures effective control of the airflow pattern. Turbulence air is directed around the impeller atomizing device. 3 is a rotary atomizing device or nozzle atomizing device. 4 shows a connecting pipe system made of stainless steel, which can be easily removed for cleaning. 5 are steps to the entrance to the upper chamber area. 6 is the switch for an air valve to activate the pneumatic lifting device when the chamber lid is lifted. 7 is a highly effective stainless steel cyclone, where the powder and spent drying air are separated. 8th is a glass jar where powder is recovered. 9 is a centrally located instrument panel. 10 is a centrifugal exhaust fan with three-phase motor. 11 is an attenuator for controlling the air flow and 12 is an electric air heater that delivers drying air temperatures up to 350 ° C. The drying air temperature can be adjusted continuously using a timer. The maximum energy consumption is 7.5 kW. Evaporation capacity

Weight and dimensions Weight 280 kg length 1800 mm height 2200 mm depth 925 mm

Power supply. The unit can only be connected to a three-phase mains connection (50 or 60 Hz) at alternating voltages of 440, 415, 400, 380, 220, 220 V. operate.

All with the liquid or parts in contact with the product are made of acid-resistant stainless steel Made of steel AISI 316.

Claims (12)

Pharmaceutically acceptable hollow microcapsules in the form of a dry powder, wherein more than 30% of the microcapsules have a diameter within a range of 2 μm and at least 90% have a diameter in the range of 1.0-8.0 μm. Pharmaceutically acceptable hollow microcapsules in the Form of a dry powder, the interquartile range of Diameter 2 μm or less and the median diameter is between 2.0 μm and 8.0 μm inclusive. Pharmaceutically acceptable hollow microcapsules after Claim 1 or claim 2, having proteinaceous walls, wherein at least 90% of the microcapsules have a wall thickness of 40-500 nm and at least 50% (w / w) of the protein in the walls of the microcapsules so cross-linked are that they resist extraction in 1% HCl for 2 minutes. Pharmaceutically acceptable hollow microcapsules after Claim 3 wherein at least 95% of the protein cross-links as indicated are. Pharmaceutically acceptable hollow microcapsules according to claim 1 or claim 2, wherein at least 10% of the microcapsules, when suspended in water, apply a pressure of 2.66 x 10 ⁴ Pa for 0.25 s without bursting, collapsing or filling with water can survive. Microcapsules according to any one of claims 1 to 5, which are sterile. Method, which is the stage of sputtering a solution or dispersion of a proteinaceous wall-forming material in a liquid carrier in a gas to form pharmaceutically acceptable hollow microcapsules by evaporation of the liquid carrier includes. The method of claim 7, wherein the thus obtained Product of the further step of reducing water solubility from at least the outside the microcapsules is subjected. The method of claim 7 or 8, wherein the wall-forming Material is collagen, gelatin or serum albumin. The method of claim 9, wherein in the step of Claim 7, the protein solution or dispersion as indicated, with discrete microcapsules a diameter of 0.01-50.0 microns formed become. Method according to one of claims 8 to 10, wherein the conditions the stage of claim 7 are such that the stage of claim 8 is achieved substantially simultaneously. Microcapsules produced by a process according to a the claims 7 to 11 available are.